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J. Biol. Chem., Vol. 275, Issue 31, 24215-24221, August 4, 2000

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Characterization of Isolated Acidocalcisomes of Trypanosoma cruzi^*

David A. Scott and Roberto Docampo

From the Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802

Received for publication, March 23, 2000, and in revised form, May 15, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The acidocalcisome is an acidic calcium store in trypanosomatids with a vacuolar-type proton-pumping pyrophosphatase (V-H⁺-PPase) located in its membrane. In this paper, we describe a new method using iodixanol density gradients for purification of the acidocalcisome from Trypanosoma cruzi epimastigotes. Pyrophosphatase assays indicated that the isolated organelle was at least 60-fold purified compared with the large organelle (10,000 × g) fraction. Assays for other organelles generally indicated no enrichment in the acidocalcisome fraction; glycosomes were concentrated 5-fold. Vanadate-sensitive ATP-driven Ca²⁺ uptake (Ca²⁺-ATPase) activity was detectable in the isolated acidocalcisome, but ionophore experiments indicated that it was not acidic. However, when pyrophosphate was added, the organelle acidified, and the rate of Ca²⁺ uptake increased. Use of the indicator Oxonol VI showed that V-H⁺-PPase activity generated a membrane potential. Use of sulfate or nitrate in place of chloride in the assay buffer did not affect V-H⁺-PPase activity, but there was less activity with gluconate. Organelle acidification was countered by the chloride/proton symport cycloprogidiosin. No vacuolar H⁺-ATPase activity was detectable in isolated acidocalcisomes. However, immunoblots showed the presence of at least a membrane-bound V-H⁺-ATPase subunit, while experiments employing permeabilized epimastigotes suggested that vacuolar H⁺-ATPase and V-H⁺-PPase activities are present in the same Ca²⁺-containing compartment.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chagas disease remains an important world health problem. Advances are being made in parts of South America in blocking transmission from insect vectors or blood transfusion (1), but more effective chemotherapy is needed for the millions who are already infected. This is especially true for treatment of the long term chronic phase of the disease (2, 3). The rational development of new drugs depends on the identification of differences between human metabolism and that of the causative parasite, Trypanosoma cruzi.

An unusual feature of T. cruzi, in comparison with mammalian cells, is the storage of calcium in acidic organelles, which we termed acidocalcisomes (4). Initially identified in permeabilized cells (4), we subsequently isolated these organelles (5, 6). We found that they had a high density (6); had a high content of calcium, magnesium, sodium, and zinc (5); and contained pyrophosphate and polyphosphates (7). Such are the characteristics of inclusions found in various microorganisms over many years, which have been known as volutin granules or by a variety of other names (8). However, despite being analyzed in situ, especially by x-ray dispersion microanalysis (5, 9, 10), these organelles have not previously been isolated and characterized with respect to their enzymatic and transport activities. Our results to date have already produced a novel finding, namely that the acidocalcisome possesses vacuolar-type proton-pumping pyrophosphatase (V-H⁺-PPase)¹ activity (6). Until recently, this activity had been definitively shown to be present only in the vacuoles and plasma membranes of plants (11, 12) and in the photosynthetic membranes of Rhodospirillum rubrum, where it acts as a pyrophosphate synthase (13). V-H⁺-PPases have now been found in other bacteria and an archaeon (14), and we have demonstrated activity in other trypanosomatids (15, 16) and in malarial parasites (17) and Toxoplasma,² but, beyond one report (18), their presence in animals is undocumented. We have recently cloned, sequenced, and expressed the V-H⁺-PPase gene from T. cruzi.³

The purpose of the present work was to further characterize the isolated T. cruzi acidocalcisome, particularly with respect to features that had been attributed to this compartment in permeabilized cell experiments. We examined ATP-driven Ca²⁺ uptake and, by the use of ionophores, tested whether Ca²⁺ was accumulated into an acidic environment, as found in digitonin-permeabilized T. cruzi (4). We checked for the establishment of a membrane potential in the acidocalcisome by the action of the V-H⁺-PPase and the effect of different anions and the chloride/proton symport cycloprogidiosin (CPG; Ref. 19) on proton uptake. We searched for the presence of Na⁺/H⁺ exchange activity, which we had previously found in acidocalcisome fractions from Trypanosoma brucei (16). Finally, we looked for the presence of H⁺-ATPases in the acidocalcisome, since our initial work implicated a vacuolar-type H⁺-ATPase in the acidification of this organelle (4), and we have also before found evidence for the presence of a P-type H⁺-ATPase in internal membranes of T. cruzi (20). The present work was facilitated by the use of a new isolation procedure, in which Percoll was replaced as the density gradient substrate by iodixanol.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Leupeptin, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, N-p-tosyl-L-lysine chloromethyl ketone, ATP, Oxonol VI, arsenazo III, ionophores except ionomycin, and reagents for marker enzyme assays were purchased from Sigma. Silicon carbide (400 mesh) was bought from Aldrich. Bafilomycin A₁ was purchased from Kamiya Biomedical (Thousand Oaks, CA). 4-(2-Aminoethyl)benzenesulfonyl fluoride and ionomycin (free acid) were from Calbiochem. Pepstatin was from Roche Molecular Biochemicals. Iodixanol (40% solution (OptiPrep), Nycomed) and Dulbecco's PBS were obtained from Life Technologies. Aminomethylenediphosphonate (AMDP) (21) and a polyclonal antiserum that had been raised against a keyhole limpet hemocyanin-conjugated synthetic peptide corresponding to the hydrophilic loop IV (antibody 324 or PAB_TK) of plant V-H⁺-PPase (22) were kindly provided by Prof. Philip Rea (University of Pennsylvania). CPG (19) was a gift of Prof. Hajime Hirata (Himeji Institute of Technology, Hyogo, Japan). Mouse monoclonal antibody N2 against Dictyostelium V-H⁺-ATPase 100-kDa subunit was bought from the Monoclonal Antibody Center of the University of Hawaii. A rabbit antiserum was raised against a recombinant nonconserved portion of a cloned T. cruzi P-type H⁺-ATPase and affinity-purified.⁴ Secondary antisera, molecular weight markers, and Coomassie Blue protein assay reagent were from Bio-Rad. EnzChek phosphate assay kit was from Molecular Probes, Inc. (Eugene, OR). The enhanced chemiluminescence detection kit was bought from Amersham Pharmacia Biotech. All other reagents were analytical grade.

Isolation of Acidocalcisomes-- Epimastigotes (~2 × 10¹⁰) of the Y strain of T. cruzi were grown as described previously (4), collected by centrifugation, and washed twice in Dulbecco's PBS and once in lysis buffer (125 mM sucrose, 50 mM KCl, 4 mM MgCl₂, 0.5 mM EDTA, 20 mM K-Hepes, 5 mM dithiothreitol, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 µM pepstatin, 10 µM leupeptin, 10 µM trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, and 10 µM N-p-tosyl-L-lysine chloromethyl ketone, pH 7.2). The cell pellet was mixed with 1.5× wet weight silicon carbide and lysed by grinding with a pestle and mortar for 60 s. The lysate was clarified first by centrifugation at 144 × g for 5 min and then 325 × g for 10 min. The second pellet was washed under the same conditions, and the supernatant fractions were combined and centrifuged for 30 min at 10,500 × g. The pellet was resuspended in 4 ml of lysis buffer with the aid of a 22-gauge needle and applied to a discontinuous gradient of iodixanol, with 4-ml steps of 24, 28, 34, 37, and 40% iodixanol, diluted in lysis buffer. The gradient was centrifuged at 50,000 × g in a Beckman SW 28 rotor for 60 min. The acidocalcisome fraction pelleted on the bottom of the tube and was resuspended in lysis buffer.

H⁺ Transport Assay and Membrane Potential Measurement-- Pyrophosphate-driven H⁺ uptake into acidocalcisomes was assayed using acridine orange as described before (6), except that the standard buffer used was 120 mM KCl, 2 mM MgCl₂, 50 mM K-Hepes, 50 µM EGTA, pH 7.2. In assays for H⁺-ATPase activity, pyrophosphate (0.1 mM) was replaced by ATP (1 mM). In the alternate buffers used, the chloride salts were replaced with sulfates, nitrates, or gluconates. The membrane potential induced in acidocalcisomes by H⁺-PPase activity was monitored using Oxonol VI (16), with the same buffer used for the assay of H⁺ transport.

Ca²⁺ Uptake Assays-- The uptake of Ca²⁺ into organelles within digitonin-permeabilized epimastigotes was assayed using the Ca²⁺ binding dye arsenazo III. A previous assay method (6) was used, except that EGTA and calcium chloride were not added to the assay mixture; the only free Ca²⁺ in the assay came from the cells or was contaminant in the buffer used (typically 3-11 µM). Oligomycin and antimycin A were also omitted from the assay buffer, since there was no evidence of mitochondrial uptake under these conditions. Experiments with isolated acidocalcisomes were performed in the same manner, except that whole cells were replaced by the purified fraction, and digitonin was omitted.

Enzyme Assays-- PPase was assayed by measuring released phosphate using the EnzChek phosphate assay kit as described before (6) with the microtiter plate modification (16). The sensitivity of this method to phosphate was calibrated in the different buffers used. Hexokinase (glycososomal marker) and -mannosidase (lysosome) were assayed as before (6, 16). Alanine and aspartate aminotransferases, which have dual mitochondrial/cytosolic locations in T. cruzi (23), were assayed by a modification of a previous method (24). For alanine aminotransferase, a 0.1-ml mixture of 50 mM Na-Hepes, pH 7.2, 4 mM 2-oxoglutarate, 10 mM L-alanine, 2 units/ml lactate dehydrogenase, and 0.2 mM NADH was added to sample in a microtiter well, and the activity was recorded at 340 nm and 30 °C in a PowerWave 340i plate reader (Bio-tek Instruments). For aspartate aminotransferase, the same method was used, with the substitution (at the same concentrations) of L-aspartate for alanine and malate dehydrogenase for lactate dehydrogenase.

Immunoblot Methods-- Proteins were separated by SDS-PAGE, using 4-15% Ready Gels (Bio-Rad), and blotted onto nitrocellulose (NitroPure, MSI, Westborough, MA) with a Bio-Rad Mini Transblot apparatus by standard techniques. Subsequent processing steps were done in Dulbecco's PBS containing 0.1% Tween 20. Blots were blocked for 1 h in 5% nonfat dry milk, washed three times, and incubated with primary antibody, diluted as per the Fig. 2 legend, for 1 h at room temperature. Blots were then washed three times, incubated for 30 min with horseradish peroxidase-labeled anti-rabbit IgG (1:10,000) or anti-mouse IgG (1:3000) as appropriate, washed three times, and processed for chemiluminescence detection as per the manufacturer's (Amersham Pharmacia Biotech) instructions. Photographic exposures of 10 s to 4 min were made. Molecular weights were calculated using prestained molecular weight markers. Before reprobing with other antibodies, blots were stripped for 30 min at 50 °C in 62.5 mM Tris-HCl, pH 6.8, containing 2% SDS and 1% 2-mercaptoethanol, washed three times in PBS/Tween 20, and reblocked as above. Spot intensity was measured with an Alpha Imager 2000 imaging system (Alpha Innotech Corp., San Leandro, CA).

Fluorescence and Electron Microscopy-- CPG (100 nM) was added to T. cruzi epimastigotes resuspended in Dulbecco's PBS at 4 × 10⁷ cells/ml and incubated at room temperature for 5 min. Cells were collected by centrifugation and washed twice in PBS before observation with an Olympus BX-60 fluorescence microscope fitted with a red emission filter. Digital images were recorded as described before (25). Electron microscopy of acidocalcisome fractions dried onto sample grids or fixed and sectioned was done as before (5, 6).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of Acidocalcisomes Using Iodixanol-- The utility of the method described here for the purification of acidocalcisomes was assessed by assaying marker enzymes and comparing the activities in the large organelle fraction applied to the iodixanol gradient and the acidocalcisome fraction from the base of the gradient (Table I). Pyrophosphatase was assessed as the pyrophosphate hydrolytic activity sensitive to the specific V-H⁺-PPase inhibitor AMDP (6, 21). Its yield was 10%, whereas the yield of protein was only 0.16%, a 62-fold purification. This may actually be a large underestimate of the degree of purification of the acidocalcisome, since the H⁺-PPase, although a marker for the acidocalcisome, also appears to be located on the cell surface (6). The only other organelle that was purified to any extent in the acidocalcisome preparation was the glycosome, evidenced by a 5-fold purification of hexokinase. Lysosomes (marked by -mannosidase; Refs. 5 and 6) were not enriched in this fraction. For the mitochondrion, succinate cytochrome c reductase (5, 6) and citrate synthase (23) assays were unusable because of dithiothreitol interference. Instead, we assayed alanine and aspartate aminotransferases, which are, respectively, 60% cytosolic/40% mitochondrial, and 10% cytosolic/90% mitochondrial (23). Neither of these activities was purified in the acidocalcisome fraction. The acidocalcisome was therefore enriched at least 10-fold more than these other cell compartments by this technique.

Electron microscopy of the acidocalcisome fraction, either by observation of air-dried samples (Fig. 1A) or of samples fixed and sectioned (Fig. 1B) had the same appearance as the acidocalcisome fraction from Percoll gradients (6, 7). We found before that acidocalcisomes have very varied diameters even in intact cells (5, 6), and, when fixed, they lose their electron-dense content to a variable extent, which results in a heterogeneous appearance (6, 7).

View larger version (58K): [in this window] [in a new window]   Fig. 1.   Electron microscopy of the acidocalcisome fraction prepared by the iodixanol procedure. A, unfixed and unstained acidocalcisomes air-dried directly onto microscopy grids. B, fixed and sectioned acidocalcisome fraction. Scale bars, 1 µm.

Immunoblot Analysis of H⁺ Pumps-- We assessed the acidocalcisome fraction for the presence of different H⁺ pumps using antibodies. First, we used an antiserum raised against a peptide, 15 amino acids in length, from the Arabidopsis H⁺-PPase, which we previously found to recognize the T. cruzi H⁺-PPase (6). The corresponding peptide is present in the T. cruzi sequence with only a single conservative substitution.³ As expected, the antiserum detected a band of the appropriate size in the acidocalcisome, even when as little as 0.3 µg of protein was loaded onto the gel, but the H⁺-PPase was not detected in 3 µg of the 10,000 × g fractions (Fig. 2A, top). The same blot was reprobed with an antiserum raised against a P-type H⁺-ATPase, which has been found to be mainly located on the cell surface of T. cruzi but is also present on some internal membranes⁴ (Fig. 2A, bottom). The 10,000 × g pellet fraction gave a strong response, estimated by densitometry to be 3 times as strong as the response to the acidocalcisome fraction on an equivalent protein basis. In other words, the purification between these fractions of this H⁺-ATPase was <1×. This indicates that the acidocalcisome fraction was not enriched in plasma membrane.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Immunoblots of acidocalcisome fractions using antibodies against H+ pumps. Various amounts (shown in terms of µg of protein above each lane) of 10,000 × g supernatant (S) or pellet (P) fractions and purified acidocalcisomes (AC) were loaded onto gels. On the right is shown calculated molecular mass for the indicated bands, in kDa. A, top, reaction with antiserum raised against a conserved V-H+-PPase peptide, diluted 1:1000; bottom, the same blot reprobed with affinity-purified antiserum raised against T. cruzi P-type H+-ATPase, 1:10,000. The two lanes indicated for each fraction were loaded with samples obtained from two different acidocalcisome preparations. B, reaction with monoclonal antibody N2 raised against the 100-kDa subunit of Dictyostelium V-H+-ATPase, 1:50. One acidocalcisome preparation was compared with different amounts of the 10,000 × g fractions from the same preparation.

Work with permeabilized T. cruzi had previously suggested the presence of a vacuolar H⁺-ATPase in acidocalcisomes (4), and a monoclonal antibody (N2), raised against the 100-kDa subunit of Dictyostelium vacuolar H⁺-ATPase (26), had proven to be immunoreactive against vacuoles in T. cruzi (25). We found that this antibody recognized a band slightly larger than the expected molecular weight in isolated acidocalcisomes (Fig. 2B), but only when a large amount of sample (13 µg of protein) was loaded. Comparison with varied amounts of the 10,000 × g pellet fraction on the blot indicated that this band was less than 4-fold purified in the acidocalcisome.

H⁺-PPase Activity of Acidocalcisomes: Anion Effects-- H⁺-PPase activity was detectable in the purified acidocalcisome fraction using acridine orange (Fig. 3). In this assay, the weak base acridine orange accumulates and dimerizes in vesicles as they acidify, leading to changes in its spectral properties (27). Vesicle acidification induced by pyrophosphate may be measured as the decrease in absorbance at 493-530 nm (6). Activity varied between 0.014 and 0.095 absorbance units/min/µg of protein in the standard assay (nine preparations). Replacement of chloride in the buffer by sulfate or nitrate did not affect the activity, whereas gluconate led to lower proton uptake rates (Fig. 3). In experiments with different preparations, sulfate gave 94 ± 3% of the initial rate with chloride (n = 3 experiments), nitrate gave 98 ± 10% of the chloride rate (n = 4), and gluconate gave 61 ± 20% of the chloride rate (n = 5). These anion effects were also tested in pyrophosphate hydrolysis (phosphate detection) assays. These assays included the proton ionophore nigericin to dissipate proton or membrane potential gradients across the acidocalcisome membrane. Here, the respective activities in the different buffers in comparison with the chloride buffer were as follows: sulfate, 95 ± 41%; nitrate, 107 ± 10%; gluconate, 89 ± 11% (n = 3 in all cases; the excessive variation in the activity with sulfate was due to the phosphate assay system being much less sensitive in the presence of sulfate). These results suggest that there is a requirement for anion transport to balance H⁺ uptake during H⁺-PPase activity and that chloride, sulfate, or nitrate can fulfill this requirement, but gluconate is transported less efficiently, if at all.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Pyrophosphate-induced acidification of acidocalcisomes in the presence of different anions, measured as acridine orange uptake. Anion in buffer was as follows: gluconate (a), sulfate (b), chloride (c), nitrate (d). In the experiment shown, 0.16 µg of acidocalcisome protein was added per ml of assay medium. Pyrophosphate (PPi, 0.1 mM) was added where indicated.

Membrane Potential of Acidocalcisomes-- Using the above assay conditions (chloride buffer), but with replacement of acridine orange by Oxonol VI, it was possible to detect a membrane potential () following pyrophosphate addition (Fig. 4). The sharp rise in absorbance (A₆₃₀-A₅₉₆) upon pyrophosphate addition indicated the establishment of an inside-positive membrane potential (28). This slowly drifted down over the following several minutes (Fig. 4, trace a). In contrast, when 1 µM carbonyl cyanide m-chlorophenyl hydrazone was added shortly after pyrophosphate, the absorbance dropped sharply (trace b), indicating a dissipation of the membrane potential by this proton ionophore. The addition of 20 µM AMDP before pyrophosphate (trace c) substantially inhibited the absorbance increase, implicating H⁺-PPase activity in the establishment of the membrane potential. The results shown are representative of four experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Pyrophosphate-induced membrane potential in acidocalcisomes, measured by increase in Oxonol VI absorbance. Reaction mixture contained the standard assay buffer plus 1 µM Oxonol VI and 0.64 µg/ml acidocalcisome protein. Pyrophosphate (PPi, 0.1 mM) was added at the point shown. In trace b, carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1 µM) was added where indicated, and in trace c, 20 µM AMDP was added prior to the start of the experiment.

Cycloprogidiosin Dissipates Proton Gradients of Acidocalcisomes-- CPG is a compound isolated from a marine bacterium that has been shown to uncouple H⁺-PPase activity. This effect occurs in chloride but not sulfate buffers; therefore, CPG appears to act as a chloride/proton symport (19). Against the H⁺-PPase activity of isolated acidocalcisomes, we found that, in the standard chloride buffer, 10 nM CPG collapsed PP_i-induced proton gradients almost as effectively as the proton/potassium ionophore nigericin (Fig. 5A). However, where sulfate was substituted for chloride, CPG had little effect (Fig. 5B). CPG has also been shown to be accumulated in vacuoles of the malaria parasite Plasmodium falciparum (against which it is a potent growth inhibitor; Ref. 29), and treatment of live epimastigotes of T. cruzi with 100 nM CPG (Fig. 5C) or 10 nM (not shown) stained vacuoles within the cells, which were observable using a fluorescence microscope.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Effects of CPG on acidocalcisome pyrophosphate-driven proton uptake and accumulation of the dye in T. cruzi epimastigotes. A, H+-PPase activity, assayed as in Fig. 3, in the standard chloride assay buffer. Additions are as marked: 0.1 mM pyrophosphate (PPi); 10 nM CPG; 10 µM nigericin (Nig). B, H+-PPase activity, assayed in sulfate buffer. Additions as in A. C, fluorescence of live epimastigotes labeled with 100 nM CPG and viewed through a red emission filter (left). Phase-contrast images of the same cells are shown on the right. Scale bar, 10 µm.

Ca²⁺ Uptake by Acidocalcisomes-- Uptake of Ca²⁺ by acidocalcisomes was demonstrable using the calcium indicator arsenazo III (Fig. 6, trace a). The addition of ATP led to uptake of Ca²⁺ (shown by a decrease in absorbance at 675-685 nm indicating reduction in medium Ca²⁺). This uptake was reduced by 76 ± 4% (mean ± S.D. in three experiments) upon the addition of 100 µM o-vanadate, indicating the operation of a P-type Ca²⁺-ATPase, as found before in permeabilized cells (4). The addition of ionomycin released accumulated Ca²⁺, and the release rate was little affected by the further addition of nigericin. This showed that, as isolated, the acidocalcisome fraction was not acidic, since ionomycin alone cannot mobilize Ca²⁺ out of an acidic organelle but first requires the pH gradient across the organelle membrane to be dissipated by a proton ionophore like nigericin or a weak base such as ammonium chloride (4, 30). However, as noted above (Fig. 3), the isolated organelle can be acidified with pyrophosphate. When 0.1 mM pyrophosphate was added prior to ATP in Ca²⁺ uptake experiments (Fig. 6, trace b), there was an initial sharp drop in absorbance due to Ca²⁺ chelation and then a steady rise as pyrophosphate was hydrolyzed and chelated Ca²⁺ was released. Despite this rising background, when ATP was added, the Ca²⁺ uptake rate was 26 ± 4% (mean ± S.D. in three experiments) faster than without prior pyrophosphate addition. The addition of 100 µM vanadate led to a rise in the medium Ca²⁺ at a rate similar to that pre-ATP. This rate was not affected by ionomycin but was accelerated greatly by the addition of nigericin, confirming that the Ca²⁺ was contained in an organelle that could be acidified by pyrophosphate, i.e. the acidocalcisome.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   ATP-mediated uptake of Ca2+ by isolated acidocalcisomes, measured with the Ca2+-binding dye Arsenazo III. A decrease in absorbance indicates decreasing medium Ca2+ or increasing vesicular Ca2+. Assay mixtures contained 125 mM sucrose, 65 mM KCl, 2 mM MgCl2, 20 mM K-Hepes, pH 7.2, 40 µM Arsenazo III, and acidocalcisome fraction (0.5 µg of protein/ml). ATP (1 mM), sodium o-vanadate (Van, 100 µM), ionomycin (Ion, 1 µM), and nigericin (Nig, 2 µM) were added at the points indicated, plus, in trace b only, pyrophosphate (PPi, 0.1 mM).

Lack of H⁺-ATPase and Na⁺/H⁺ Exchange Activity in Acidocalcisomes-- No H⁺-ATPase activity was detectable in the acidocalcisome fraction isolated as above, using the acridine orange assay and 1 mM ATP, in assays of 11 separate preparations (detection limit <1% of the activity obtained with 0.1 mM pyrophosphate). These included preparations where the lysis buffer contained either 50 mM DTT or 20 mM sodium dithionite as reducing agent in place of 5 mM DTT. The activity of vacuolar H⁺-ATPases has been shown to depend on the reduction of critical cysteine residues (31), and the Neurospora vacuolar H⁺-ATPase was shown to be optimally active in the presence of 20 mM dithionite (32).

Na⁺/H⁺ exchange activity, found before in isolated acidocalcisomes of T. brucei (16), could not be detected in six separate preparations of T. cruzi acidocalcisomes. This activity was inferred before from the release of vesicular acridine orange, accumulated as the result of H⁺-ATPase or H⁺-PPase activity (16, 33). In the latter case, the addition of ADP was necessary to stimulate the activity (16). We could not detect any acridine orange release, with or without 1 mM ADP, by up to 80 mM NaCl, whereas as little as 6 mM NaCl was found before to release acridine orange from acidocalcisomes in permeabilized T. brucei (33). Alternative assay buffers used before in Na⁺/H⁺ exchange experiments (16, 33) were also tried. We also grew T. cruzi epimastigotes in SDM-79 medium, which was the medium used before for culture of T. brucei procyclic forms (16, 33), and Leishmania donovani promastigotes (15), in which exchange activity was detected. Finally, we grew T. cruzi in the standard brain heart infusion medium (4), supplemented with 0.25 M NaCl (which was the maximum amount of the salt that permitted normal growth; the normal medium contains approximately 0.1 M NaCl). None of these strategies yielded detectable exchange activity.

Involvement of H⁺-ATPase in Ca²⁺ Uptake-- The above data indicate an absence of H⁺-ATPase activity and the near absence of H⁺-ATPase proteins from the acidocalcisome. Previously, however, we presented evidence for a link between H⁺-ATPase and Ca²⁺ uptake, albeit one that was founded on the finding that Ca²⁺ dissipated ATP-generated H⁺ gradients, which might be explained by inhibition of H⁺-ATPase activity rather than uptake of Ca²⁺ (and exchange with H⁺) into an acidic compartment (4). To investigate this link further, we performed ATP-driven calcium uptake experiments in permeabilized epimastigotes in the presence of the specific V-H⁺-ATPase inhibitor bafilomycin A₁ (34). The addition of 40 nM bafilomycin A₁, a concentration just sufficient to completely inhibit T. cruzi V-H⁺-ATPase activity (20) but ineffective against T. cruzi V-H⁺-PPase activity (6), reduced the rate of ATP-driven Ca²⁺ uptake by 27% (Fig. 7, traces a and e; Table II). The addition of 100 µM vanadate inhibited the Ca²⁺ uptake rate by 71% (trace d and Table II), and the addition of bafilomycin plus vanadate only increased the inhibition slightly (Table II), implying that the Ca²⁺ uptake was largely the result of a vanadate-sensitive Ca²⁺-ATPase and not a Ca²⁺/H⁺ exchanger driven by the V-H⁺-ATPase. Our results suggest that Ca²⁺-ATPase activity is enhanced by acidification of the interior of the compartment in which it is located by a V-H⁺-ATPase. Ca²⁺-ATPases of different types in mammalian cells have been shown to exchange Ca²⁺ for H⁺ (35, 36).

View larger version (31K): [in this window] [in a new window]   Fig. 7.   ATP-mediated uptake of Ca2+ by permeabilized T. cruzi epimastigotes. Assays were as described in the legend to Fig. 6, with the addition of whole epimastigotes (0.18 mg/ml protein), creatine kinase (5 units/ml), and creatine phosphate (2 mM) as an ATP-regenerating system and 20 µM digitonin (added 4 min before ATP). Experimental runs contained, in addition, 40 nM bafilomycin A1 (trace a), 0.1 mM pyrophosphate (trace b), 40 nM bafilomycin A1 plus 0.1 mM pyrophosphate (trace c), 0.1 mM sodium o-vanadate (trace d). Trace e, control. ATP (1 mM) was added at zero time, and ionomycin (Ion, 1 µM) and nigericin (Nig, 2 µM) were added at the points indicated.

To check whether V-H⁺-PPase was also present in this compartment, pyrophosphate was added in addition to ATP, with or without bafilomycin A₁ (Fig. 7, traces b and c; Table II). The addition of pyrophosphate at 10 or 100 µM did not enhance Ca²⁺ uptake, but in the presence of pyrophosphate at either concentration, the effects of bafilomycin were almost exactly canceled out, implying that both proton pumps (V-H⁺-ATPase and V-H⁺-PPase) were present in the same compartment and that, when the former was inhibited, the latter could fulfill the role of enhancing Ca²⁺-ATPase activity. (Pyrophosphate at 10 µM was used, since the H⁺-PPase is as active at this concentration as at 100 µM,⁵ but problems with Ca²⁺ chelation (as noted above) are reduced.) The effects of the ionophores ionomycin and nigericin supported these results. Following bafilomycin treatment, most of the releasable Ca²⁺ was released by ionomycin alone (i.e. the Ca²⁺-containing compartment was deacidified; Fig. 7, trace a), but, with the addition of pyrophosphate, most of the Ca²⁺ was released only after the further addition of nigericin, irrespective of whether bafilomycin was present or not (i.e. pyrophosphate maintained the acidity of the Ca²⁺-containing compartment; Fig. 7, traces b and c).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We previously used a Percoll-based method for the separation of acidocalcisomes from various trypanosomatid species (6, 15, 16). This had a big disadvantage, in that the Percoll precipitated at the bottom of the centrifuge tube along with the acidocalcisome pellet. Attempts to wash out the Percoll resulted in large losses of acidocalcisome material. Therefore, we switched to using iodixanol, a soluble density gradient material that did not precipitate and could therefore be removed completely at the end of the centrifugation run, allowing the acidocalcisome pellet to be resuspended in the buffer of choice. The current protocol affords a substantial purification of the acidocalcisome compared with the large organelle (10,000 × g) fraction, at least 60-fold as marked by H⁺-PPase activity (Table I). This may be a substantial underestimate, given that an unknown fraction of the H⁺-PPase resides on the cell surface (6). It is notable how little protein the purified fraction contains, only 0.2% relative to the large organelle fraction. Nevertheless, at least 10 bands were observable by Coomassie Blue-stained SDS-PAGE of 0.5 µg of acidocalcisome protein (result not shown).

H⁺-PPase activity generates a membrane potential across the acidocalcisome membrane, as measured with Oxonol VI (Fig. 4). This potential peaks quickly and then slowly declines, either through cation efflux or anion uptake. Investigation of anion requirements to balance H⁺-PPase activity (Fig. 3) showed that there was a requirement, which could not be fulfilled by gluconate, but there was no stringent requirement for chloride. The activity with nitrate may be explicable by diffusion of this anion through the membrane (37); sulfate, in contrast, is less membrane-permeable than chloride (37). Therefore, the acidocalcisome membrane may have an anion channel generally permeable to small anions.

Results with CPG supported previous findings that this compound acts as a H⁺/Cl symporter (19), since it collapsed pyrophosphate-generated H⁺ gradients, but only in chloride buffer and not sulfate buffer (Fig. 5). This implies that anions are freely exchanged across the acidocalcisome membrane, as the chloride is being transported out of, rather than into, the organelle. CPG has been shown to have antimalarial properties in vitro and in vivo (29) but may also be immunosuppressive (38), which could limit its use as an antiparasitic agent.

The isolated acidocalcisome accumulated Ca²⁺ when treated with ATP (Fig. 6). That this was due to a Ca²⁺-ATPase, and not a Ca²⁺/H⁺ exchanger driven by a pH gradient, was evidenced, first, by o-vanadate inhibition, and second, by the complete release of Ca²⁺ by ionomycin (no extra release with nigericin addition) in the absence of pyrophosphate, implying that, as isolated, the organelle is not acidic. The enhancement of the Ca²⁺ uptake rate with pyrophosphate treatment implies that the Ca²⁺-ATPase is a Ca²⁺/H⁺-exchanging ATPase.

No H⁺-ATPase activity could be found in the isolated acidocalcisome, whereas H⁺-ATPase and H⁺-PPase activities were apparently located in the same Ca²⁺-containing compartment in permeabilized epimastigotes, as demonstrated by the counterbalance of bafilomycin and pyrophosphate effects. The most conservative explanation, without invoking the presence of other acidic calcium storage compartments, is that the Ca²⁺ compartment in permeabilized cells is indeed the acidocalcisome. It is possible that the low levels of H⁺-ATPase protein detected by immunoblot in the acidocalcisome are active in permeabilized cells but that the H⁺-ATPase complex dissociates, losing its peripheral subunits, or otherwise becomes inactive during purification, as has been observed in other cases (31, 39). The monoclonal antibody used for the immunoblots was raised against a membrane subunit of the H⁺-ATPase (26) and would therefore detect the V-H⁺-ATPase even if it had lost its peripheral subunits. The acidocalcisome is a small organelle, with an average diameter of 200 nm in epimastigotes (5), and may therefore need only a few active H⁺-ATPase complexes to acidify. Vacuolar H⁺-ATPase protein has been detected in acidocalcisome-like vacuoles in cryosectioned T. cruzi by electron microscopy using both the monoclonal antibody employed here (25) and a polyclonal antiserum against the whole V-H⁺-ATPase complex (40).

Alternatively, there may be two populations of acidocalcisomes in these cells, which differ in their density and in the possession or lack of a V-H⁺-ATPase, and it is the dense, H⁺-ATPase-lacking acidocalcisome that is isolated by the protocol described in this paper. This would be in agreement with previous co-localization studies by immunofluorescence of intact cells (25), where not all vacuoles showing reaction with antibodies against the acidocalcisomal Ca²⁺-ATPase reacted with antibodies against the V-H⁺-ATPase.

The absence of detectable Na⁺/H⁺ exchange activity despite the use of different approaches that might induce it shows that the acidocalcisome is not a fixed entity across trypanosomatid species. The Na⁺/H⁺ exchange activity is readily detected in T. brucei and L. donovani (15, 33) and has been demonstrated in isolated acidocalcisomes of T. brucei (16). Further differences may be expected in the analogous organelles found in other microorganisms (8), so there remains much to be explored in the biochemistry of these cellular compartments.

	ACKNOWLEDGEMENTS

We thank Philip Rea and Yolanda Drozdowicz for anti-V-H⁺-PPase antiserum and AMDP, and we thank Hajime Hirata for CPG. Affinity-purified anti-P-type H⁺-ATPase antiserum was prepared by Wen Yan. Linda Brown cultured many liters of T. cruzi epimastigotes. Claudia Rodrigues provided helpful comments on the manuscript and assistance with electron microscopy.

	FOOTNOTES

^* This work was supported by the National Institutes of Health Grant AI-23259 (to R. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Ave., Urbana, IL 61802. Tel.: 217-333-4856; Fax: 217-244-7421; E-mail: d-scott1@uiuc.edu.

Published, JBC Papers in Press, May 17, 2000, DOI 10.1074/jbc.M002454200

² Rodrigues, C. O., Scott, D. A., Bailey, B. N., de Souza, W., Benchimol, M., Moreno, B., Urbina, J. A., Oldfield, E., and Moreno, S. N. J. (2000) Biochem. J. 349, in press.

³ J. E. Hill, D. A. Scott, S. Luo, and R. Docampo, submitted for publication.

⁴ S. Luo, W. Yan, H.-G. Lu, and R. Docampo, manuscript in preparation.

⁵ D. A. Scott, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: V-H⁺-PPase, vacuolar-type proton-pumping pyrophosphatase; H⁺-PPase, proton-pumping pyrophosphatase; V-H⁺-ATPase, vacuolar-type proton-ATPase; H⁺-ATPase, proton-ATPase; AMDP, aminomethylenediphosphonate; CPG, cycloprogidiosin; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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